Coelenterazine and vargulin are naturally-occurring imidazopyrazinone luciferins used in bioluminescence imaging, but their scarcity and need to supply them exogenously to cell culture and animal models limit their use. I propose to elucidate the biosynthesis of these compounds in bioluminescent zooplankton. In Specific Aim 1, I discuss the possible biosynthetic origins for these molecules, and my plans to identify the genes coding for the requisite biosynthetic enzymes. In Specific Aim 2, I describe how I would apply these newly-discovered genes to economically produce coelenterazine by fermentation in S. cerevisiae, addressing a critical barrier to progress. In Specific Aim 3, I propose initial experiments to assess whether the ability to effect the luciferin?s biosynthesis in human cell culture model improves bioluminescence imaging contrast. Successful completion of this project could have revolutionary impacts on biological imaging, akin to how development of Green Fluorescent Protein from a biological curiosity into a multipurpose biochemical marker has radically transformed the way we apply microscopy and imaging to biological questions. Besides improving imaging signal-to-noise, localized in situ luciferin production in cell culture could aid research into inter- and intracellular signaling. Moreover, expression of the luciferin biosynthetic machinery in animals would allow for long-term noninvasive in vivo bioluminescence experiments such as gene expression monitoring and assessing tumor burden in cancer disease models.